Optical imaging techniques have been shown to be useful diagnostic tools for a number of biomedical applications. Increasingly, the structure of biological tissue is being studied using the interaction of the tissue with light. For example, using light scattering spectroscopy (LSS) the size and index of refraction of scatterers comparable in size to the wavelength of light are profiled by measuring the periodicity of the spectra of reflected light. In standard pathological analysis, sensitivity to variations in the size and index of refraction of cell nuclei can give indications of abnormal cell activity. For example, pre-cancerous epithelial cells exhibit nuclear enlargement.
Generally, obtaining spectroscopic information from tissue in vivo is difficult as light scattered from surrounding tissues often obscures the desired optical signal. This can be overcome through a combination of spectroscopy with a biomedical imaging techniques which provide the ability to isolate the light scattered from a specific portion of a biological sample. For example, light scattering techniques have been used during endoscopic procedures to measure the size distribution of cell nuclei and refractive index changes in the epithelial linings of the body. In these studies, the intensity of white light back-scattered from the tissue is collected via an optical fiber probe and spectrally analyzed. The cell nuclei behave like Mie scatterers. Such particles exhibit periodic intensity variations with wavelength that are proportional to their sizes (typically 5-15 μm) and relative refractive indices. Thus light scattering spectroscopy (LSS) is of interest because changes in the size of cell nuclei and their chromatin content (related to refractive index) are primary indicators of dysplasia, the precursor of cancer, and treatment is most simple and effective when implemented at this early stage. However, intensity based LSS only provides a two-dimensional image.
Optical Coherence Tomography (OCT) is another technique that has been developed as a diagnostic tool for the study of biological tissue especially for noninvasive cross-sectional imaging in biological systems. OCT uses low-coherence interferometry to produce a three-dimensional image of optical scattering from internal tissue microstructures in a way that is analogous to ultrasonic pulse-echo imaging. OCT has longitudinal and lateral spatial resolutions of a few micrometers and can detect reflected signals as small as about 10−10 of the incident optical power.
In OCT, tissue is placed in a Michelson interferometer illuminated by a broadband light source. Due to the limited coherence length of the source (typically 10-15 μm), light returning from the interferometer reference arm and light backscattered by internal sample reflections interferes constructively or destructively only when the interferometer arm optical path lengths are matched to within the source coherence length. Scanning the reference arm length generates a localized interference pattern to appear in the detector current for every internal sample reflection as a function of depth along the sample arm beam. A sample containing many reflection sites distributed along its depth (such as biological tissue) generates a detector current that contains the sum of multiple, overlapping copies of this interference pattern. A map of tissue reflectivity versus depth (called an A-Scan) is obtained by scanning the reference mirror while recording the detector current. The envelope, or outline of the detector current, may be recorded with high dynamic range by scanning the reference mirror at fixed velocity, while demodulating the detector current at the resulting Doppler frequency. Cross-sectional images of tissue backscatter (called B-Scans) are acquired by collecting sequential A-scans while scanning the probe beam across the tissue surface. The resulting two-dimensional datasets are plotted as gray-scale or false-color images. However, OCT does not provide a functional image as the resultant resolution does not provide an image of the cell nuclei, whose size, shape and other characteristics can reveal abnormal cell activity.